DNA mismatch repair (MMR) exemplified by the E. coli methyl-directed MMR pathway, targets base pair mismatches that arise through DNA replication errors, homologous recombination and DNA damage. Inactivation of MMR results in a large increase in the rate of spontaneous mutation and is associated with both sporadic and hereditary cancers. In addition to its role in post-replication repair, components of the MMR pathway also influence the expansion of trinucleotide repeats associated with syndromes such as Huntingtons disease, play an essential role in assuring the proper pairing of chromosomes during meiosis, modulate homologous recombination involving closely related sequences, and participate in the generation of antibody diversity at immunoglobulin gene loci. A key step in mismatch repair is the recognition of DNA mispairs by MMR proteins and the licensing of excision repair. This is a critical problem in cells because the gapped DNA intermediate of MMR is easily converted into lethal double-strand breaks if not efficiently repaired. We are examining how human MMR proteins MutSalpha (hMSH2-MSH6), MutSbeta (hMSH2-hMSH3), and MutLalpha (hMLH1-hPMS2) interact with each other at the sites of DNA mismatches and how nucleotide cofactors modulate such interactions and license downstream excision steps. In collaboration with Dr. Dorothy Erie, we are using atomic force microscopy (AFM) to examine the conformations of individual protein-DNA complexes that are formed during MMR. A focus is the recognition of mismatches in DNA that result from DNA damage, e.g. alkylated guanines, by wild type MMR proteins and those harboring specific mutations found in HNPCC patients. We have developed in vitro assays to monitor mismatch repair and are mapping excision tracts by the mutant proteins. These studies have important implications for understanding how this mismatch repair pathway contributes to genome stability and cancer avoidance.